Alzheimer's, cystic fibrosis, infectious diseases, and cancer all have a common feature: failure to efficiently destroy incorrectly folded or assembled proteins. Transmembrane and secreted proteins use a signal peptide (SP) to direct translation to the endoplasmic reticulum (ER), where an error-prone process of protein folding and modification occur. The ER-associated degradation (ERAD) process identifies misfolded proteins, leading to addition of ubiquitin chains using E1, E2, and E3 enzymes. Polyubiquitinated proteins then are extracted from the ER membrane, a process known as retrotranslocation or dislocation, and delivered to cytosolic proteasomes for degradation. Many pathogens, including viruses and bacteria, exploit ERAD to further their dissemination. Mouse mammary tumor virus (MMTV) is a complex retrovirus that uses ERAD for replication and induction of breast cancer and leukemia. MMTV produces a regulatory protein (Rem), which is directed to the ER by a long signal peptide (Rem-SP) containing all the functional motifs found in HIV Rev. Rem is cleaved by signal peptidase in the ER lumen to yield N-terminal Rem-SP and a unique C-terminus (RemCT). In an unprecedented pathway, Rem-SP is retrotranslocated to the cytosol, where it avoids proteasomal degradation prior to nuclear import. Rem-SP then binds viral RNA to allow efficient nuclear export and expression. Although retrotranslocation is believed to require polyubiquitination of target proteins, our preliminary data indicate that the E enzyme used for all known ERAD substrates is dispensable for Rem-SP retrotranslocation. Our hypothesis is that Rem-SP uses a previously unknown process that does not require polyubiquitination or the E1 enzyme Uba1 for retrotranslocation. In the first specific aim, two approaches will be used to identify cellular components involved in subversion of ERAD. C-terminal GFP tagging of Rem blocks retrotranslocation and will be used to purify a retrotranslocation intermediate and associated cellular proteins for analysis by mass spectrometry. An alternative approach will use a fluorescence-based reporter assay and a small-hairpin library to identify new cellular proteins involved in ERAD and Rem trafficking. In the second specific aim, exciting preliminary data have been presented showing that MMTV proviruses lacking RemCT expression have many G to A mutations compared to wild-type proviruses. Co-expression of Rem and activation-induced cytidine deaminase (AID), a known mutagen required for antibody somatic hypermutation and class switch recombination, results in AID degradation. We propose that MMTV is the first virus to antagonize AID to avoid both viral genome mutation and maturation of the MMTV-specific antibody response. This idea will be tested by monitoring RemCT localization and its ability to antagonize cellular cytidine deaminases both in cell culture and in knockout mice in vivo. Further understanding of ERAD and intrinsic immunity are essential to develop new treatments for cancer and pathogenic viral infections. PUBLIC HEALTH RELEVANCE: Endoplasmic reticulum-associated degradation (ERAD) is essential for destruction of many defective cellular proteins found in infectious diseases, Alzheimer's disease, cystic fibrosis, and cancer, but is subverted by a number of pathogens, including the oncogenic retrovirus mouse mammary tumor virus (MMTV). The MMTV-encoded Rem protein uses ERAD to control viral protein expression and appears to be the first viral protein that antagonizes the activation-induced cytidine deaminase (AID) protein, which causes double- stranded DNA breaks and human cancers. Studies of ERAD and intrinsic immunity and their exploitation by pathogens are essential for development of new and effective treatments for many human diseases.